General western blot 
    protocol 
     
    Guidance for running an efficient and 
    accurate experiment 
     
     
                
                             
                             
       General western      Contents 
       blot protocol 
        
                           ‒   Introduction 
                           ‒   Solution and reagents 
                           ‒   Sample lysis 
                           ‒   Sample preparation 
                           ‒   Loading and running the gel 
                           ‒   Antibody staining 
                           ‒   Useful links
                            Introduction 
                            Western blotting is used to visualize proteins that have been separated by gel 
                            electrophoresis. The gel is placed next to a nitrocellulose or PVDF (polyvinylidene 
                            fluoride) membrane and an electrical current causes the proteins to migrate from the 
                            gel to the membrane. The membrane can then be probed by antibodies specific for 
                            the target of interest, and visualized using secondary antibodies and detection 
                            reagents.
                            Solutions and reagents 
                            Lysis buffers 
                             
                            These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C 
                            for up to a year. 
                            NP-40 buffer 
                            ‒   150 mM NaCl  
                            ‒   1.0% NP-40 (possible to substitute with 0.1% Triton X-100)  
                            ‒   50 mM Tris-HCl, pH 8.0  
                            ‒   Protease inhibitors 
                            RIPA buffer (radioimmunoprecipitation assay buffer) 
                            ‒   150 mM NaCl  
                            ‒   1.0% NP-40 or 0.1% Triton X-100  
                            ‒   0.5% sodium deoxycholate  
                            ‒   0.1% SDS (sodium dodecyl sulphate)  
                            ‒   50 mM Tris-HCl, pH 8.0  
                            ‒   Protease inhibitors 
                             
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                                      Tris-HCl 
                                      ‒   20 mM Tris-HCl  
                                      ‒   Protease inhibitors 
                                      Running, transfer and blocking buffers 
                                       
                                      Laemmli 2X buffer/loading buffer 
                                      ‒   4% SDS  
                                      ‒   10% 2-mercaptoethanol  
                                      ‒   20% glycerol  
                                      ‒   0.004% bromophenol blue  
                                      ‒   0.125 M Tris-HCl 
                                       
                                      Check the pH and adjust to 6.8 
                                      Running buffer (Tris-Glycine/SDS) 
                                      ‒   25 mM Tris base  
                                      ‒   190 mM glycine  
                                      ‒   0.1% SDS 
                                       
                                      Check the pH and adjust to 8.3 
                                      Transfer buffer (wet) 
                                      ‒   25 mM Tris base  
                                      ‒   190 mM glycine  
                                      ‒   20% methanol 
                                      ‒   Check the pH and adjust to 8.3 
                                           
                                      For proteins larger than 80 kDa, we recommend that SDS is included at a final 
                                      concentration of 0.1%. 
                                      Transfer buffer (semi-dry) 
                                      ‒   48 mM Tris  
                                      ‒   39 mM glycine  
                                      ‒   20% methanol  
                                      ‒   0.04% SDS 
                                      Blocking buffer 
                                      3–5% milk or BSA (bovine serum albumin) 
                                      Add to TBST buffer. Mix well and filter. Failure to filter can lead to spotting, where tiny 
                                      dark grains will contaminate the blot during color development. 
                                       
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        General western       Sample lysis 
        blot protocol 
         
                              Preparation of lysate from cell culture 
                              1.  Place the cell culture dish on ice and wash the cells with ice-cold PBS. 
                                   
                              2.  Aspirate the PBS, then add ice-cold lysis buffer (1 mL per 107 cells/100 mm dish/150 
                                     2                     6                        2
                                  cm flask; 0.5 mL per 5x10  cells/60 mm dish/75 cm  flask). 
                                   
                              3.  Scrape adherent cells off the dish using a cold plastic cell scraper, then gently 
                                  transfer the cell suspension into a pre-cooled microcentrifuge tube. Alternatively 
                                  cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer in 
                                  a microcentrifuge tube. 
                                   
                              4.  Maintain constant agitation for 30 min at 4°C. 
                                   
                              5.  Centrifuge in a microcentrifuge at 4°C. You may have to vary the centrifugation 
                                  force and time depending on the cell type; a guideline is 20 min at 12,000 rpm but 
                                  this must be determined for your experiment (leukocytes need very light 
                                  centrifugation). 
                                   
                              6.  Gently remove the tubes from the centrifuge and place on ice, aspirate the 
                                  supernatant and place in a fresh tube kept on ice, and discard the pellet. 
                              Preparation of lysate from tissues 
                              1.  Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as 
                                  possible to prevent degradation by proteases. 
                                   
                              2.  Place the tissue in round-bottom microcentrifuge tubes or Eppendorf tubes and 
                                  immerse in liquid nitrogen to snap freeze. Store samples at -80°C for later use or 
                                  keep on ice for immediate homogenization. For a ~5 mg piece of tissue, add ~300 
                                  μL of ice cold lysis buffer rapidly to the tube, homogenize with an electric 
                                  homogenizer, rinse the blade twice with another 2 x 200 μL lysis buffer, then 
                                  maintain constant agitation for 2 h at 4°C (eg place on an orbital shaker in the 
                                  fridge). Volumes of lysis buffer must be determined in relation to the amount of 
                                  tissue present; protein extract should not be too dilute to avoid loss of protein and 
                                  large volumes of samples to be loaded onto gels. The minimum concentration is 
                                  0.1 mg/mL, optimal concentration is 1–5 mg/mL. 
                                   
                              3.  Centrifuge for 20 min at 12,000 rpm at 4°C in a microcentrifuge. Gently remove 
                                  the tubes from the centrifuge and place on ice, aspirate the supernatant and 
                                  place in a fresh tube kept on ice; discard the pellet. 
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